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ORIGINAL RESEARCH COMMUNICATION |
1 From the Division of Pathology (KDRS, BEW, LN-Z, and NMB), the Department of Gastroenterology and Nutrition (JEH), and the Department of Pediatrics (SJC and CH), Cincinnati Children’s Hospital Medical Center, and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH (KDRS and JEH); the Department of Gastroenterology and Hepatology, University of Perugia, Perugia, Italy (CC and DC); Sanitarium Development and Innovation, Cooranbong, Australia (SJC and CH); the Department of Physiology and Developmental Biology and The Neuroscience Center, Brigham Young University, Provo, UT (EDL); and the Department of Biomedical Sciences, Colorado State University, Fort Collins, CO (TDL and RJH)
2 These findings were presented at the 4th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, San Diego, CA, November 4-7, 2001, and at the 5th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, Orlando, FL, September 21-24, 2003. 3 Supported by grants from the National Institutes of Health (R01CA73328) and the National Center for Research Resources (RR08084). 4 Address reprint requests to KDR Setchell, Clinical Mass Spectrometry, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail: kenneth.setchell{at}cchmc.org.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: Because it was unclear which enantiomer was present in humans, our objectives were to characterize the exact structure of equol, to examine whether the S- and R-equol enantiomers are bioavailable, and to ascertain whether the differences in their conformational structure translate to significant differences in affinity for estrogen receptors.
Design: With the use of chiral-phase HPLC and mass spectrometry, equol was isolated from human urine and plasma, and its enantiomeric structure was defined. Human fecal flora were cultured in vitro and incubated with daidzein to ascertain the stereospecificity of the bacterial production of equol. The pharmacokinetics of S- and R- equol were determined in 3 healthy adults after single-bolus oral administration of both enantiomers, and the affinity of each equol enantiomer for estrogen receptors was measured.
Results: Our studies definitively establish S-equol as the exclusive product of human intestinal bacterial synthesis from soy isoflavones and also show that both enantiomers are bioavailable. S-equol has a high affinity for estrogen receptor ß (Ki = 0.73 nmol/L), whereas R-equol is relatively inactive.
Conclusions: Humans have acquired an ability to exclusively synthesize S-equol from the precursor soy isoflavone daidzein, and it is significant that, unlike R-equol, this enantiomer has a relatively high affinity for estrogen receptor ß.
Key Words: Equol • soy isoflavones • humans • pharmacokinetics • bacteria
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Equol is not of plant origin and is exclusively a product of intestinal bacterial metabolism (9), as evidenced from the finding that infants fed soy formula up to the age of 4 mo (10, 11) and germ-free rats fed soy-containing diets (12) do not make equol. When fed soy protein, a common ingredient of most commercial rodent diets (13, 14), rats and mice are prolific equol producers. In contrast, humans are unique among animals in that, for reasons that remain unclear, only 20–35% of the adult population make equol after ingesting soy foods or being challenged with pure isoflavones (3, 15, 16). Several studies have suggested that those who are equol producers show more favorable responses to soy isoflavone–containing diets (17–21), which leads to the possibility that equol is a more potent isoflavone than genistein (9), which has been so extensively studied in the last decade.
Equol, unlike its precursor daidzein or genistein, is unique in having a chiral carbon atom at position C-3 of the furan ring (Figure 1
). It therefore can occur as 2 distinct enantiomeric forms, S-equol and R-equol, which differ significantly in their conformational structure. We recently showed that S-equol is unique in that it not only possesses estrogenic properties but also is a potent antagonist of dihydrotestosterone in vivo, which has significant implications for the prevention of prostate cancer and other androgen-related conditions (22). We can find no other example of a molecule that is both a selective estrogen and an androgen antagonist. Establishing the diastereoisomerism of equol production is therefore important both in view of the possible differences in biological actions of the enantiomers and in aid of the future development of strategies either to use equol pharmacologically or to manipulate equol production in humans to enhance the effectiveness of soy diets.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Written informed consent was obtained from all subjects. The studies were approved by the Cincinnati Children’s Hospital Medical Center Institutional Review Board.
Studies of S- and R-equol bioavailability
Purified samples of S-equol and R-equol (20 mg) obtained by semipreparative chiral-phase HPLC chromatographic separation of a racemic mixture were prepared in capsules and randomly administered orally to 3 healthy adults (1 female and 2 male) on different occasions separated by a washout period of 1 wk. This study was performed by the ethical standards of the Department of Gastroenterology and Hepatology, University of Perugia, Italy, in accordance with the Helsinki Declaration of 1975, as revised in 1983. Each equol enantiomer was taken with a glass of water after an overnight fast and before eating breakfast. Blood samples (10 mL) were obtained via an indwelling catheter placed in the antecubital vein at timed intervals immediately before and then 1, 2, 3, 4, 8, 12, and 24 h after the administration of equol. These samples were centrifuged for 10 min at 2200 x g and room temperature, and the plasma was removed for the measurement of equol concentrations by stable-isotope dilution GC-MS with selected ion monitoring, as detailed below. The plasma equol concentration–time profiles for the 3 persons were ascertained by using a noncompartmental approach. The WINNONLIN computer program (version 3.0; Pharsight Corporation, Cary, NC) was used for this analysis. The total area under the plasma concentration–time curve (AUC 0
; AUCinf) was computed by using the following equation:
 | (1) |
z = apparent elimination rate constant.
The z was determined from the slope of the best-fitting regression line of the plasma samples in the terminal phase. At least 4 time points were included in the estimation of z. When required, appropriate weighting schemes (usually 1/y or 1/y2, where y is the observed plasma concentration) were used to improve the goodness of fit. The choice of the number of points included in the terminal phase of the plasma concentration-time curves was based on the weighted residual (difference between model predicted and observed concentrations) values, dispersion of the residual values, and regression coefficient. In all cases, the regression lines were drawn without exclusion of any time points, and the R2 values were >0.91. The terminal half-life was calculated as ln(2)/z; the systemic clearance after oral administration was determined as dose/AUCinf, and the apparent volume of distribution after oral administration was determined as dose/(z.AUCinf). Note that "F" used in the abbreviations of these terms (CL/F and Vz/F, respectively) refers to the bioavailable fraction after oral administration, which in this case is unknown.
Specificity of intestinal bacterial equol production
Freshly passed stool samples were obtained from 3 recognized equol producers and 3 equol nonproducers for culture of the fecal bacteria exactly as described previously (3). These samples were collected by staff of the Pathology Department of the Sydney Adventist Hospital, Australia, after informed consent was obtained. The freshly passed fecal sample (1 g) was added to 9 mL sterile distilled water and trypticase broth, to which 0.02 mg daidzein was added. The broths were incubated anerobically for 3 days at 37 °C. A 2-mL aliquot of this incubation mixture was taken, diluted with distilled water (2 mL), and centrifuged for 5 min at 2200 x g and at room temperature. The supernatant was passed through a Bond Elut C18 solid-phase cartridge to extract the isoflavones, which were recovered by elution with methanol (5 mL). This sample was subjected to chiral-phase HPLC coupled with ESI-MS to separate and identify the S- and R-equol diastereoisomers as described below. Confirmation of the identity of each enantiomeric form of equol was based on its chromatographic retention time and mass spectrum as compared with pure standards of S- and R-equol.
Animal study—identification of S-equol in rat urine
A sample of adult Sprague-Dawley rat urine was obtained from animals used in a previously published study of rodents fed commercial chow containing soy isoflavones that was performed with the approval of the Cincinnati Children’s Hospital Medical Center Animal Use Committee (14).
Analytic methods
Equol concentrations were measured in the urine and plasma by either ESI liquid chromatography–MS (ESI-LC-MS) or GC-MS techniques, or both, after liquid-solid extraction, hydrolysis of the conjugates with a mixed ß-glucuronidase and sulfatase enzyme preparation (Helix pomatia; Sigma Chemicals, St Louis, MO), reextraction, and preparation of a volatile derivative in the case of GC-MS analysis. The methods have been outlined in previous studies of isoflavones (2, 18, 28, 29). Separate quantification of the S- and R-equol was achieved by using a recently developed chiral-phase HPLC chromatographic method (described below) that resolves the S- and R- enantiomers of equol.
Determination of S- and R-equol concentrations in plasma by gas chromatography–mass spectrometry
The concentrations S- and R-equol in plasma (0.5 mL) samples collected after administration of these enantiomers were measured by GC-MS after the addition of [13C] stable isotope–labeled analogs of S- and R-equol as internal standards for quantification. These internal standards were obtained by chiral-phase HPLC chromatographic separation of (±)([13C]equol that was synthesized from [2-13C]daidzein as described elsewhere (30). Isoflavones were extracted on a solid-phase octadecylsilane-bonded silica cartridge and hydrolyzed enzymatically with a mixed ß-glucuronidase and sulfatase preparation (H. pomatia). After reextraction and purification, the t-BDMS ether derivatives were prepared, and the derivatized samples were analyzed by selected ion monitoring GC-MS as described previously (18, 27, 28).
Chiral-phase HPLC and electrospray ionization–mass spectrometry separation and identification of diastereoisomers of equol
Sample extracts for the characterization of the enantiomeric form of equol in urine and plasma were taken up in 100 µL of the HPLC mobile phase, and a 20-µL sample was injected on column. Separation of the enantiomers of equol was performed on a Chiralcel column (Chiral Technologie, Inc, Exton, PA). The mobile phase used to enable baseline resolution of S- and R-equol was a gradient elution beginning with 90% hexane and 10% ethanol (by vol) and linearly increasing to a final composition of 10% hexane and 90% ethanol (by vol) over a 15-min period at a flow rate of 1 mL/min. Detection of S- and R- equol is possible by ultraviolet absorption at 260 nm, even though equol has poor ultraviolet absorption characteristics, provided relatively high concentrations are injected on column. This wavelength was monitored to establish the conditions using the pure standards of equol. However, for detecting equol enantiomers in human plasma and urine, the greater sensitivity of ESI-MS was necessary. ESI-MS was performed on a Micromass Quattro LC/MS (Waters Corp, Milford, MA). The HPLC effluent to the ESI probe was split 10:1. The desolvation temperature was 300 °C, and the source temperature was 100 °C. The sampling cone was held at 50 V and the extractor at 2 V. Data were collected in the negative ion mode, and the [M-H]– ions monitored were a mass-to-charge ratio (m/z) of 241 (equol) and a m/z of 242 ([2-13C]equol). The identity of the enantiomeric form of equol in the human plasma and urine samples was based on the retention time of the eluting peak in the mass chromatogram compared with the mass chromatograms obtained for pure standards of S- and R-equol analyzed under identical conditions.
Studies of estrogen receptor binding
Synthesis of hormone receptor proteins
Full-length human estrogen receptor (ER) 
[(ER) pcDNA-ER; RH Price, University of California, San Francisco] and rat ERß (pcDNA-ERß; TA Brown, Pfizer, Groton, CT) expression vectors were used to synthesize hormone receptors in vitro by using the transcription and translation–coupled reticulocyte lysate system (Promega, Madison, WI) with T7-RNA polymerase, during a 90-min reaction at 30 °C. Translation reaction mixtures were stored at –80 °C until further use.
Saturation isotherms
To calculate and establish the binding affinity of the R and S equol enantiomers for ER and ERß, 100-µL aliquots of reticulocyte lysate supernatant were incubated at optimal time and temperature—90 min at room temperature (ERß) and 18 h at 4 °C (ER)—with increasing (0.01–100 nmol/L) concentrations of [3H]17ß-estradiol (E2). These times were determined empirically, and they represent optimal binding of receptor with estrogen. Nonspecific binding was assessed by using a 300-fold excess of the ER agonist diethylstilbestrol in parallel tubes. After incubation, bound and unbound [3H]E2 was separated by passing the incubation reaction through a 1-mL lipophilic Sephadex LH-20 column (Sigma-Aldrich Co, St Louis, MO). Columns were constructed by packing a disposable pipette tip (1 mL; Labcraft, Curtin Matheson Scientific, Inc, Houston, TX) with TEGMD (10 mmol Tris-Cl/L, 1.5 mmol EDTA/L, 10% glycerol, 25 mmol molybdate/L, and 1 mmol dithiothreitol/L; pH 7.4)-saturated Sephadex according to previously published protocols (31, 32). For chromatography, the columns were reequilibrated with TEGMD (100 µL), and the incubation reactions were added individually to each column and allowed to incubate on the column for an additional 30 min. After this incubation, 600 µL TEGMD was added to each column, flow-through was collected, 4 mL of scintillation fluid was added, and samples were counted (5 min each) in a 2900 TR Packard scintillation counter (Packard Bioscience, Meriden, CT).
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RESULTS |
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. S-equol eluted from the chiral-phase column with a shorter retention time than that of R-equol, and baseline resolution was achieved. The identity of both enantiomers was confirmed by isolation of both peaks and measurement of optical dichroism. Also shown in Figure 2 is a typical HPLC-ESI-MS mass chromatogram of the negative ion m/z 241 corresponding to the pseudomolecular ion ([M-H]–) of equol obtained from the analysis of hydrolyzed extracts of human urine. All samples of human urine analyzed were found to contain a single equol enantiomer with a retention time (6.75 min) that exactly corresponded to the retention time of the pure standard of S-equol (ie, 6.77 min). There was no evidence for the presence of R-equol (which elutes from the HPLC column with a significantly longer retention time of 7.51 min) in any sample of human urine when analyzed under the same chromatographic conditions. Likewise, a sample of urine from a rat, a species that is exclusively an equol producer (12, 14), was found, on the basis of its retention index, to have exclusively the S-equol enantiomer (Figure 2). Similar findings were obtained from the analysis of a selection of samples (n = 10) of human plasma collected from equol producers who had consumed 2 x 240 mL soymilk. Consistent with the urinary analysis, the ESI-MS profiles of plasma showed one major peak in all samples analyzed, and this had a retention time corresponding to that of the S-equol standard.
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. The administration of both enantiomers yielded similar plasma pharmacokinetic profiles, which confirmed that the 2 diastereoisomers are similarly bioavailable. Equol rapidly appeared in plasma and disappeared with a terminal elimination half-life of 4.9 ± 1.6 and 6.2 ± 0.2 h, respectively, for the S- and R-enantiomers, and there was no obvious difference between these values. A comparison of the computed pharmacokinetics of the diastereoisomers is shown in Table 1. There were no statistical differences in maximum plasma concentration, time to reach maximum plasma concentration, terminal elimination half-life, AUCinf, apparent volume of distribution, and systemic clearance between R- and S-equol, and no difference in the absorption rates of the 2 enantiomers.
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. For comparison, the mass chromatograms of a pure mixture of S- and R- equol are also shown, and, on the basis of the retention time, only R-equol was found in the plasma. After administration of S-equol, ESI-MS confirmed that S-equol appeared unchanged in the 2-h plasma sample (data not shown). These data provided evidence that both enantiomeric forms of equol were absorbed without change and that no racemization occurred during or after intestinal uptake. Finding only the S-equol enantiomer in human urine and plasma suggested that this must be the result of the exclusive bacterial production of S-equol in the intestine.
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. The cultured fecal flora from the equol producers showed a peak corresponding to equol that had a retention time identical to that of the authentic pure standard of S-equol. By contrast, the supernatant fluid from an equol nonproducer showed negligible production of S-equol. These results establish conclusively that fecal bacteria are selective in producing only S-equol as the principal metabolite of the soy isoflavone, daidzein.
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(Ki = 6.41 ± 1 nmol/L) was relatively poor. In contrast, R-equol possessed only 4.8% and 25.0% as much relative binding affinity, respectively, for ERß (Ki = 15.4 ± 1.3 nmol/L) and for ER (Ki = 27.38 ± 3.8 nmol/L) as did S-equol. For comparison, 17ß-estradiol binds ER with a Kd of 0.13 nmol and ERß with a Kd of 0.15 nmol. S-equol thus shows ER selectivity with a high affinity for ERß, whereas R-equol can, at best, be classified as a weak estrogen. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the uterus, it is almost certain that these early reports of binding affinities refer to equol’s binding to ER, rather than to ERß. Using preparations of recombinant steroid receptors, we have shown that only the S enantiomer of equol binds to ERs with sufficient affinity to be of physiologic relevance based on circulating equol concentrations typically found in humans (9). Furthermore, almost 50% of equol circulates unbound to serum protein (39), whereas endogenous estrogen is >95% protein bound. This protein-binding status of equol is likely to enhance its biological potency because it is only the "free" or unbound fraction that is available for receptor occupancy. The relative binding affinity of the R- and S-equol enantiomers for ER were 0.47% and 2.0% with that of 17ß-estradiol. However, S-equol is largely ERß selective and has a relatively high affinity for this receptor subtype. S-equol binds ERß 
20% with as much affinity as does 17ß-estradiol (equol: Ki = 0.7 nmol/L; 17ß-estradiol: Kd = 0.15 nmol/L), whereas the R enantiomer bound at 1% of the affinity. These findings are corroborated by several studies (40–42) that also show equol to have selective affinity for ERß, and, therefore, equol can be defined as a type of selective ER modulator.
As a potent antagonist of dihydrotestosterone (DHT) in vivo, equol is also unique, in that we can find no other example of a compound that has selective estrogen action and yet also has the ability to be an antagonist of androgen action (22). It is interesting that the mechanism of its anti-androgen action differs from that of the anti-androgen drugs used in clinical practice to block the effects of DHT. For example, equol has no affinity for the androgen receptor (22) and therefore does not function as an androgen receptor blocker. It also does not appear to alter the synthesis of DHT in the way that 5-reductase inhibitors do, but, rather, it appears to bind directly to DHT (22), and this effect is seen with both R- and S-equol (TD Lund, RJ Handa, ED Lephart, KDR Setchell, unpublished data, 2003).
Given the distinctly different biological actions of the diastereoisomers of equol, particularly with regard to their affinity toward ERs, it is relevant to define the stereoisomerism of equol production in humans. Using ESI-MS with selected ion monitoring, we analyzed urine and plasma samples from healthy adults, and by our ability to separate the 2 diastereoisomers with the use of chiral-phase column chromatography, we have shown for the first time that S-equol is the only enantiomer circulating in human blood and excreted in urine (Figures 2
and 3). This is also true in the rat, a species that is predominantly an equol producer (14). The logical explanation for the finding of a single enantiomer in plasma and urine is that intestinal bacteria are stereoselective in their synthesis, but the possibility that both enantiomers would be made in the intestine, but only one, the S enantiomer, would be absorbed required addressing. Furthermore, racemization of R-equol to S-equol during the former’s absorption was an alternative possibility that was feasible and required investigation.
The separate oral administration of pure S- and R-equol to 3 healthy adults clearly showed that both enantiomers, when present in the intestine, are efficiently absorbed and appear rapidly in plasma. There were no differences in the pharmacokinetics of the 2 enantiomers. The bioavailability of equol as measured by the dose-adjusted AUCinf is relatively high when compared with the bioavailability of genistein and daidzein reported in previous studies (18, 27, 28). The clearance rate of equol was also much slower than that of the soy isoflavones, which contributes to the maintenance of high circulating equol concentrations observed in rodents (14). ESI-MS established that, after its administration, R-equol appeared in plasma unchanged, and therefore the possibility of bacterial production of R-equol in the intestine with racemization to S-equol during absorption can be confidently excluded. Thus, these data taken together are indicative of the enantiomer-specific production of S-equol by intestinal bacteria. This is now confirmed by in vitro experiments in which human fecal flora from equol producers were cultured under anerobic conditions and incubated with daidzein or soy isoflavones. After 72 h in culture, S-equol was the sole enantiomer identified in the supernatant. Thus, given that humans, rats, and sheep all produce S-equol—and it is likely that macaque monkeys (43), chimpanzees (44), dogs (45), domestic fowl (46), cows (47), and mice (14) also do so—it is evident that the bacteria responsible for equol production are all highly selective in performing an asymmetric synthesis with production of the one enantiomer that shows the highest ligand affinity for ERß.
The formation of equol from its precursor daidzein proceeds through an intermediate, dihydrodaidzein. Our pharmacokinetic studies show that equol is rapidly absorbed from the intestine, but its formation after the initial intake of daidzein or of soy foods containing daidzin or daidzein is a time-dependent process. It generally takes >12 h for equol to appear in the plasma, and, in some adults, it may not appear for 36 h, which indicates that the colon is the site of its formation (18, 28, 48). Identification of the bacteria responsible for equol production has thus far been elusive. It is apparent that there is more than one bacterium involved because we have observed cases in which dihydrodaidzein is present in urine in the absence of equol, which is consistent with partial conversion of daidzein to equol (KDR Setchell, unpublished observations, 1995). Furthermore, in vitro incubation of fecal homogenates from some adults was shown to produce dihydrodaidzein and O-desmethylangolensin but not equol, whereas recently it was shown that some antibiotics, such as rifampicin and kanamycin, may inhibit the production of equol but not of dihydrodaidzein (6). In contrast, kanamycin virtually eliminated equol production in the plasma of cynomologus monkeys (49), which highlights the complexity of the bacterial production of equol. Attempts to identify the species of bacteria involved in equol production have yielded some information regarding strains that are capable of hydrolyzing the ß-glucoside of daidzin (50, 51) or of converting daidzein to dihydrodaidzein (52), and one report claimed that Streptococcus intermedius spp, Ruminococcus productus spp, and Bacteroides ovatus in vitro perform the required conversion (53).
In view of the apparent advantages of being an equol producer (9, 17, 19–21, 54), the question of whether it is possible to manipulate the intestinal milieu in favor of equol production when soy foods are given is relevant. Early studies by Setchell and Cassidy (55) using an in vitro model of human colonic fermentation showed that, with a background of a high nonstarch polysaccharide environment, which affords increased colonic fermentation, the conversion of daidzein to equol is complete, but no conversion occurs under low-carbohydrate conditions. This in vitro observation is supported by data from a study of 24 healthy adults showing that good equol producers were associated with a diet that was lower in saturated fat and higher in total carbohydrate (16). The role of carbohydrate in equol production, also reported by Lampe et al (15), suggests that there are important prebiotic or probiotic factors that may influence equol formation in adults. The addition of fructooligosaccharides to the diet of mice has been shown to yield higher equol concentrations (56), as did feeding potato starch (57), and, in the former study, higher equol concentrations were associated with a greater effect on bone density in this animal model. This has also been shown to be the case in a 2-y clinical study of the effectiveness of soy foods in preventing bone loss in postmenopausal women (20), in which equol producers showed a significant increase in lumbar spine bone mineral density. Whether probiotic or prebiotic diets can influence the metabolism of soy isoflavones to enhance equol production in humans is unclear (58–60), but, if not, the potential benefits of equol, with its selective ER modulator properties and its antiandrogen actions, could be optimized by the use of pure enantiomeric forms of equol as a supplement or nutraceutical.
In conclusion, our studies described here definitively establish S-equol as the only enantiomer found in human plasma and urine, and we show for the first time that this exclusivity is not due to differences in the bioavailability or metabolism of S- and R-equol but rather to the fact that intestinal microflora show enantiomeric specificity in their production of equol. These findings are of immense clinical relevance because we also found that S-equol, but not R-equol, has a relatively high affinity for ERß and is in fact a more potent estrogen than is estradiol; these findings have been corroborated by others (40–42). The significance of these findings is that humans have acquired intestinal microflora that perform an asymmetric synthesis of the only diastereoisomer of equol that has affinity for the ER and thus has the greatest potential for physiologic effects.
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ACKNOWLEDGMENTS |
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 D. C.S. Talbot, R. M. Ogborne, T. Dadd, H. Adlercreutz, G. Barnard, S. Bugel, F. Kohen, S. Marlin, J. Piron, A. Cassidy, et al. Monoclonal Antibody-Based Time-Resolved Fluorescence Immunoassays for Daidzein, Genistein, and Equol in Blood and Urine: Application to the Isoheart Intervention Study Clin. Chem., April 1, 2007; 53(4): 748 - 756. [Abstract] [Full Text] [PDF]
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 S. Joy, R. C. M. Siow, D. J. Rowlands, M. Becker, A. W. Wyatt, P. I. Aaronson, C. W. Coen, I. Kallo, R. Jacob, and G. E. Mann The Isoflavone Equol Mediates Rapid Vascular Relaxation: Ca2+-INDEPENDENT ACTIVATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE/Hsp90 INVOLVING ERK1/2 AND Akt PHOSPHORYLATION IN HUMAN ENDOTHELIAL CELL J. Biol. Chem., September 15, 2006; 281(37): 27335 - 27345. [Abstract] [Full Text] [PDF]
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 C. E. Wood, S. E. Appt, T. B. Clarkson, A. A. Franke, C. J. Lees, D. R. Doerge, and J. M. Cline Effects of High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys Biol Reprod, September 1, 2006; 75(3): 477 - 486. [Abstract] [Full Text] [PDF]
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 S. Widyarini, D. Domanski, N. Painter, and V. E. Reeve Estrogen receptor signaling protects against immune suppression by UV radiation exposure PNAS, August 22, 2006; 103(34): 12837 - 12842. [Abstract] [Full Text] [PDF]
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K. D. R. Setchell and S. J. Cole Method of Defining Equol-Producer Status and Its Frequency among Vegetarians J. Nutr., August 1, 2006; 136(8): 2188 - 2193. [Abstract] [Full Text] [PDF]
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 Y. H. Ju, J. Fultz, K. F. Allred, D. R. Doerge, and W. G. Helferich Effects of dietary daidzein and its metabolite, equol, at physiological concentrations on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice Carcinogenesis, April 1, 2006; 27(4): 856 - 863. [Abstract] [Full Text] [PDF]
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 C. E. Rufer, H. Glatt, and S. E. Kulling STRUCTURAL ELUCIDATION OF HYDROXYLATED METABOLITES OF THE ISOFLAVAN EQUOL BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY Drug Metab. Dispos., January 1, 2006; 34(1): 51 - 60. [Abstract] [Full Text] [PDF]
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R. Handayani, L. Rice, Y. Cui, T. A. Medrano, V. G. Samedi, H. V. Baker, N. J. Szabo, and K. T. Shiverick Soy Isoflavones Alter Expression of Genes Associated with Cancer Progression, Including Interleukin-8, in Androgen-Independent PC-3 Human Prostate Cancer Cells J. Nutr., January 1, 2006; 136(1): 75 - 82. [Abstract] [Full Text] [PDF]
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